Lack of adiponectin promotes formation of cholesterol gallstones in mice

Plasma adiponectin levels are reduced in obese people, and hypoadiponectinemia is recently reported to associate with cholesterol gallstone formation in human. The aim of this study was to examine the role of adiponectin in gallstone formation using adiponectin knockout mice. We analyzed male knockout and C57BL6J mice fed normal or lithogenic diet for 6 weeks. On lithogenic diet, the prevalence rate of gallstone was significantly greater in knockout mice than C57BL6J mice. The molar percentages of β and ω-muricholic acid were significantly higher and hepatic sterol 12α-hydroxylase expression (cyp8b1) was significantly lower in knockout mice than C57BL6J mice fed normal diet. The bile apolipoprotein A-I protein levels were decreased in knockout mice. Histological examination showed gallbladder wall thickening and accumulation of glycoprotein in the gallbladder of knockout mice. Gallbladder phospholipase A2-IVA expression was significantly higher in knockout mice than in C57BL6J mice fed lithogenic diet. Our results indicate that lack of adiponectin promotes gallstone formation in mice.     

Validation and application of a 96-well format solid-phase extraction and liquid chromatography-tandem mass spectrometry method for the quantitation of digoxin in human plasma

To evaluate the pharmacokinetics of digoxin in humans, a sensitive and specific LC/MS/MS method was developed and validated for the determination of digoxin concentrations in human plasma. The method was shown to be more sensitive, specific, accurate, and reproducible than common techniques such as RIA. For detection, a LC/MS/MS system with electro spray ionization tandem mass spectrometry in the positive ion-multiple reaction-monitoring (MRM) mode was used to monitor precursor to product ions of m/z 798.5-51.5 for digoxin and m/z 782.5-35.5 for the internal standard, digitoxin. The method was validated over a concentration range of 0.02-5 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 80%. Imidafenacin, coadministered in a drug-drug interaction study, had no detectable influence on the determination of digoxin in human plasma. The novel method was applied to a drug-drug interaction study of digoxin and imidafenacin and the characterization of steady-state pharmacokinetics of digoxin in humans after oral administration at a dose of 0.25 mg on days 1 and 2 followed by 0.125 mg daily doses on days 3 through 8.     

Mechanisms of resistance to spinosad in the western flower thrip, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae)

Cross‐resistance, resistance mechanisms, and mode of inheritance of spinosad resistance were studied in the western flower thrip, Frankliniella occidentalis (Pergande). Spinosad (naturalyte insecticide) showed low cross‐resistance to prothiophos (organophosphorus insecticide) and chlorphenapyr (respiratory inhibitor) showed some cross‐resistance to thiocyclam (nereistoxin). The synergists PBO (piperonyl butoxide), DEM (diethyl maleate), and DEF (s, s, s‐tributyl phosphorotrithioate) did not show any synergism on the toxicity of spinosad in the resistant strain (ICS), indicating that metabolic‐mediated detoxification was not responsible for the spinosad resistance, suggesting that spinosad may reduce sensitivity of the target site: the nicotinic acetylcholine receptor and GABA receptor. Following reciprocal crosses, dose‐response lines and dominance ratios indicated that spinosad resistance was incompletely dominant and there were no maternal effects. The results of backcross showed that spinosad resistance did not fit a single‐gene hypothesis, suggesting that resistance was influenced by several genes.     

Compensatory binding of an asparagine residue to the coordination-unsaturated type I Cu center in bilirubin oxidase mutants

Met467, the axial ligand to type I Cu in a multicopper oxidase, Myrothecium verrucaria bilirubin oxidase was substituted with a non-coordinating Phe and Leu to transform the spectral and magnetic properties and oxidase activities of the enzyme into those of fungal laccases, but the mutated type I Cu center showed properties characteristic of phytocyanins, blue copper proteins with an axial coordination of Gln, due to compensatory binding of the distal Asn459 as evidenced by a double mutation.     

Essential roles of integrin-mediated signaling for the enhancement of malignant properties of melanomas based on the expression of GD3

We reported that ganglioside GD3 enhances cell proliferation and invasion of melanomas causing stronger tyrosine-phosphorylation of p130Cas and paxillin after stimulation with fetal calf serum. Besides signals via growth factor/receptor, adhesion signals via integrin might be also enhanced by GD3. Here, roles of integrin-mediated signaling in the cell proliferation and invasion, and in the activation of adaptor molecules were examined, showing that integrin was also important for the cell growth and invasion. p130Cas and paxillin underwent stronger tyrosine-phosphorylation in GD3+ cells than in GD3− cells during the adhesion in the absence of serum. On the other hand, no proteins underwent tyrosine phosphorylation in GD3+ and GD3− cells in a suspension state when stimulated with fetal calf serum. These results suggested that integrin-mediated signaling is essential in the effects of GD3 on the malignant properties of melanomas. Co-localization of GD3 and integrin at the focal adhesion supported these results.     

Insulin induces chaperone and CHOP gene expressions in adipocytes

Adipocyte secretes bioactive proteins called adipocytokines, and biosynthesis of secretory proteins requires molecular chaperones and folding enzymes in endoplasmic reticulum (ER). ER chaperones are known to be induced by unfolded protein response (UPR) and growth factors, however, it has not been determined how ER chaperones expression is regulated in adipocytes. Here we show that insulin treatment induced GRP78 and ERO1L mRNA levels in 3T3-L1 adipocytes. Insulin also upregulated CHOP mRNA levels, but did not induce phosphorylation of eIF2α. Pretreatment with insulin protected 3T3-L1 adipocytes against thapsigargin-mediated phosphorylation of eIF2α but did not against DTT-mediated one. In vivo mice study showed that GRP78 and CHOP expressions were regulated by feeding conditions. These results suggest that insulin signaling is important to induce mRNA expressions of GRP78 and CHOP, and may have a protective role against UPR.     

DAF-16-dependent suppression of immunity during reproduction in Caenorhabditis elegans

To further understand how the nematode Caenorhabditis elegans defends itself against pathogen attack, we analyzed enhanced pathogen resistance (epr) mutants obtained from a forward genetic screen. We also examined several well-characterized sterile mutants that exhibit an Epr phenotype. We found that sterility and pathogen resistance are highly correlated and that resistance in both epr and sterile mutants is dependent on DAF-16 activity. Our data indicate that a DAF-16-dependent signaling pathway distinct from previously described pathways is involved in the activation of genes that confer resistance to bacterial pathogens. The timing of DAF-16-dependent gene activation in sterile mutants coincides with the onset of embryonic development in wild-type animals, suggesting that signals from developing embryos normally downregulate the immune response.     

Inhibitory effects of fruit juices on cytochrome P450 2C9 activity in vitro

There is limited information on the effect of fruits on human cytochrome P450 (CYP) 2C9 activity. The objective of this study was to determine the effect of fruit juice on CYP2C9-mediated drug metabolism. Nine citrus fruits and eight tropical fruits were chosen. We investigated effects of the fruits on diclofenac 4'-hydroxylation and tolbutamide hydroxylation by human liver microsomes. Among the fruits, pineapple juice showed potent inhibition of CYP2C9 activity. The addition of 25 microl (5.0% v/v) of pineapple juice resulted in almost complete inhibition. Next we examined the inhibitory effect of bromelain, a cysteine protease in pineapple. Bromelain also strongly inhibited CYP2C9 activity. In addition, E-64, a cysteine protease inhibitor, almost entirely blocked inhibition by pineapple juice and bromelain. Thus we found that pineapple juice was a potent inhibitor of CYP2C9, and that the inhibitory effect might be due to the bromelain contained in pineapple.